Human KLF1 blocking peptide

BackgroundMutations in the transcription factor, KLF1, are common within certain populations of the world. Heterozygous missense mutations in KLF1 mostly lead to benign phenotypes, but a heterozygous mutation in a DNA-binding residue (E325K in human) results in severe Congenital Dyserythropoietic Anemia type IV (CDA IV); i.e. an autosomal-dominant disorder characterized by neonatal hemolysis.ResultsTo investigate the biochemical and genetic mechanism of CDA IV, we generated murine erythroid cell lines that harbor tamoxifen-inducible (ER) versions of wild type and mutant KLF1 on a Klf1(-/-) genetic background. Nuclear translocation of wild type KLF1 results in terminal erythroid differentiation, whereas mutant KLF1 results in hemolysis without differentiation. The E to K variant binds poorly to the canonical 9bp recognition motif (NGG-GYG-KGG) genome-wide but binds at high affinity to a corrupted motif (NGG-GRG-KGG). We confirmed altered DNA-binding specificity by quantitative in vitro binding assays of recombinant zinc-finger domains. Our results are consistent with previously reported structural data of KLF-DNA interactions. We employed 4sU-RNA-seq to show that a corrupted transcriptome is a direct consequence of aberrant DNA binding.ConclusionsSince all KLF/SP family proteins bind DNA in an identical fashion, these results are likely to be generally applicable to mutations in all family members. Importantly, they explain how certain mutations in the DNA-binding domain of transcription factors can generate neomorphic functions that result in autosomal dominant disease.

Erythroid Kruppel-like factor (EKLF; KLF1) is an erythroid-specific transcription factor required for the transcription of genes that regulate erythropoiesis. In this paper, we describe the identification of a novel EKLF interactor, Ppm1b, a serine-threonine protein phosphatase that has been implicated in the attenuation of NF kappa B signaling and the regulation of Cdk9 phosphorylation status. We show that Ppm1b interacts with EKLF via its PEST1 sequence. However, its genetic regulatory role is complex. Using a promoter-reporter assay in an erythroid cell line, we show that Ppm1b superactivates EKLF at the beta-globin and BKLF promoters, dependent on intact Ppm1b phosphatase activity. Conversely, depletion of Ppm1b in CD34(+) cells leads to a higher level of endogenous beta-globin gene activation after differentiation. We also observe that Ppm1b likely has an indirect role in regulating EKLF turnover via its zinc finger domain. Together, these studies show that Ppm1b plays a multilayered role in regulating the availability and optimal activity of the EKLF protein in erythroid cells.